Mogrol glycosyltransferase and gene encoding same

ABSTRACT

To provide a mogrol glycosyltransferase and a method for producing a mogrol glycoside using the enzyme. The present invention provides a mogrol glycosyltransferase and a method for producing a mogrol glycoside using the enzyme, and a transformant into which a mogrol glycosyltransferase gene is introduced and a method for preparing the transformant.

This application is a Continuation of U.S. patent application Ser. No.15/519,326, filed Jul. 3, 2017, which is the National Stage ofInternational Patent Application No. PCT/JP2015/079907, filed Oct. 16,2015, which claims the benefit of priority of Japanese Application No.2014-213063, filed Oct. 17, 2014. The disclosures of these documents,including the specifications, drawings and claims, are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 30, 2017, isnamed P52104_SL.TXT and is 11,074 bytes in size.

TECHNICAL FIELD

The present invention relates to a protein having an activity ofsynthesizing a mogrol glycoside and a polynucleotide encoding the same;a method for producing a mogrol glycoside using the protein; atransformant highly expressing a mogrol glycosyltransferase; and amogrol glycoside prepared by the method and the use thereof.

BACKGROUND ART

The fruit of Cucurbitaceae plants contains a series of compounds calledmogrosides which are glycosides of mogrol as a triterpene phytosterol.These mogrosides have the degree of sweetness several hundred times thatof sucrose and have good quality of taste; thus, they are expected asalternative sweeteners for reduced-calorie foods like rebaudioside as aglycoside of a diterpene steviol in Asteraceae stevia.

For rebaudioside in stevia, rebaudioside A (a glycoside with 4 glucoseresidues added) and rebaudioside D (a glycoside with 5 glucose residuesadded), which are highly glycosylated, are estimated to be good in tasteand have a high degree of sweetness. Similarly, for mogrol, one with 5glucose residues added called mogroside V is good in sweetness andquality of taste and commercially available (Non Patent Literature 1).In addition, it is reported to have useful functionality other than thatas a sweetener and is a plant metabolite receiving attention (Non PatentLiterature 2). Meanwhile, it is unclear how a mogrol glycoside isbiosynthesized in a plant body such as Siraitia grosvenorii.

NON-PATENT LITERATURES

-   Non-Patent Literature 1: Kasai, R. (2008) Studies on the constituent    of Cucurbitaceous plants. YAKUGAKU ZASSGU 128(10), 1369-1382.-   Non-Patent Literature 2: Ukiya, M., et al. (2002) Inhibitory Effects    of Cucurbitane Glycosides and Other Triterpenoids from the Fruit of    Momordica grosvenori on Epstein-Barr Virus Early Antigen Induced by    Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate. J. Agric. Food.    Chem. 50, 6710-6715. (Nihon University)

DISCLOSURE OF INVENTION

As a result of conducting intensive studies, the present inventors havesucceeded in identifying an enzyme catalyzing sugar addition reaction toglucose at position 24 of a mogrol glycoside and a gene sequenceencoding the enzyme. The present invention is based on the abovefindings.

Specifically, the present invention provides the followings:

[1]

A protein selected from the group consisting of the following (a) to(c):

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 2;

(b) a protein consisting of an amino acid sequence in which 1 to 45amino acids are deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 2 and having an activity of adding asugar molecule to an R¹ group at position 24 of a compound representedby the following formula (I); and

(c) a protein having an amino acid sequence having 90% or more sequenceidentity with the amino acid sequence of SEQ ID NO: 2 and having theactivity of adding a sugar molecule to an R¹ group at position 24 of thecompound represented by the formula (I),

where R and R¹ are each independently represent H, a monosaccharide, ora disaccharide.[2]

The protein according to [1] above, wherein the sugar molecule is ahexose.

[3]

The protein according to [1] above, wherein the sugar molecule isselected from the group consisting of glucose, mannose, and galactose.

[4]

The protein according to [1] above, wherein the R and R₁ are eachindependently H or a sugar residue of glucose monomer or glucose dimer.

[5]

The method according to [1] above, wherein the compound represented bythe formula (I) is mogrol or a mogrol glycoside.

A polynucleotide selected from the group consisting of the following (a)to (e):

(a) a polynucleotide containing the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide encoding a protein consisting of the amino acidsequence of SEQ ID NO: 2;

(c) a protein consisting of an amino acid sequence in which 1 to 45amino acids are deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 2 and having an activity of adding asugar molecule to an R¹ group at position 24 of a compound representedby the following formula (I); and

(c) a protein having an amino acid sequence having 90% or more sequenceidentity with the amino acid sequence of SEQ ID NO: 2 and having theactivity of adding a sugar molecule to an R¹ group at position 24 of thecompound represented by the formula (I),

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide.[7]

The polynucleotide according to [6] above, wherein the sugar molecule isa hexose.

[8]

The polynucleotide according to [6] above, wherein the sugar molecule isselected from the group consisting of glucose, mannose, and galactose.

[9]

The polynucleotide according to [6] above, wherein the R and R¹ are eachindependently H or a sugar residue of glucose monomer or glucose dimer.

[10]

The polynucleotide according to [6] above, wherein the compoundrepresented by the formula (I) is mogrol or a mogrol glycoside.

[11]

A non-human transformant into which the polynucleotide according to [6]above is introduced.

[12]

The transformant according to [11] above, wherein the polynucleotide isone inserted into an expression vector.

[13]

The transformant according to [11] above, wherein the transformant is aplant body.

An extract of the transformant according to [11] above.

[15]

A food, a pharmaceutical product, or an industrial raw materialcomprising the extract according to [14] above.

[16]

A method for producing a protein, comprising culturing the non-humantransformant according to [11] above, wherein the protein has anactivity of adding a sugar molecule to an R¹ group at position 24 of acompound represented by the following formula (I):

where R and R₁ each independently represent H, a monosaccharide, or adisaccharide.[17]

A method for producing a mogrol glycoside, wherein the non-humantransformant according to [11] above is used.

The method according to [17] above, wherein the mogrol glycoside ismogroside IA, mogroside Ib, mogroside IE, mogroside IIA, mogroside IIA₁,mogroside IIE, mogroside III, mogroside IIIA₁, mogroside IIIA₂,mogroside IIIE, mogroside IV, mogroside IVA, mogroside V, siamenoside I,11-oxomogroside V, mogrol pentaglycoside, or a combination thereof.

[19]

A method for producing a mogrol glycoside, comprising a step of reactingthe protein according to [1] above, a UDP-sugar, and a compoundrepresented by the following formula (I):

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide.[20]

The method according to [19] above, wherein the sugar in the UDP-sugaris glucose.

The method according to [19] above, wherein the mogrol glycoside ismogroside IA, mogroside Ib, mogroside IE, mogroside IIA, mogroside IIA₁,mogroside IIE, mogroside III, mogroside IIIA₁, mogroside IIIA₂,mogroside IIIE, mogroside IV, mogroside IVA, mogroside V, siamenoside I,11-oxomogroside V, mogrol pentaglycoside, or a combination thereof.

By using the protein of the present invention and a polynucleotideencoding the protein, a mogrol glycoside (for example, mogroside V) isproduced with a high efficiency. The transformant of the presentinvention has a high content of a mogrol glycoside (for example,mogroside V); thus, the mogrol glycoside (for example, mogroside V) canbe efficiently extracted and purified from the transformant.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the formation of mogroside V as a glycosidewhere a glucose is added to mogrol by UGT enzyme.

FIG. 2 is a set of photographs showing the expression of SgUGT74G1_943protein.

FIG. 3 is an LC-MS analysis chart of a mogrol glycoside formed by theglycosylation activity of SgUGT74G1_943 protein.

FIG. 4 is a graph showing the relative activities of stevia (Sr)-derivedUGT73E1 and UGT85C2 when the activity of Siraitia grosvenorii(Sg)-derived UGT74G-943 is set to 1.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described below in detail. The followingembodiment is only illustrative for describing the present invention andis not intended to limit the invention only to the embodiment. Thepresent invention may be embodied in various forms without departingfrom its subject matter.

All references and laid-open application publications, patentpublications, and other patent literatures cited in the presentspecification are intended to be incorporated herein by reference. Thepresent specification includes the contents described in thespecification and drawings of Japanese Patent Application No.2014-213063, filed Oct. 17, 2014, on which the priority of the presentapplication is based.

The present inventors have first established that an enzyme ofCucurbitaceae Siraitia grosvenorii, SgUGT74G1_943, adds a glucosemolecule to position 24 of mogrol or a mogrol glycoside (that is, has aglycosylation activity) (FIG. 1).

The CDS sequence and the deduced amino acid sequence of SgUGT74G1_943are SEQ ID NOS: 1 and 2, respectively. The polynucleotide and the enzymecan be obtained, for example, by the technique described in Examples setforth hereinafter, a known genetic engineering technique, or a knownsynthesis technique.

1. Mogrol Glycosyltransferase

The present invention provides a protein selected from the groupconsisting of the following (a) to (c) (hereinafter referred to as“protein of the present invention”):

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 2;

(b) a protein consisting of an amino acid sequence in which 1 to 45amino acids are deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 2 and having an activity of adding asugar molecule to an R¹ group at position 24 of a compound representedby the following formula (I):

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide; and

(c) a protein having an amino acid sequence having 90% or more sequenceidentity with the amino acid sequence of SEQ ID NO: 2 and having theactivity of adding a sugar molecule to an R¹ group at position 24 of thecompound represented by the formula (I).

The protein described in (b) or (c) above is typically a variant of thenaturally occurring polypeptide of SEQ ID NO: 2; however, the proteinalso includes those artificially obtainable by using a site-directedmutagenesis method as described, for example, in “Sambrook & Russell,Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring HarborLaboratory Press 2001”, “Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons 1987-1997”, “Nuc. Acids. Res., 10, 6487(1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)”, “Gene, 34, 315(1985)”, “Nuc. Acids. Res., 13, 4431 (1985)”, or “Proc. Natl. Acad. Sci.USA, 82, 488 (1985)”.

Examples of the “protein consisting of an amino acid sequence in which 1to 45 amino acids are deleted, substituted, inserted, and/or added inthe amino acid sequence of SEQ ID NO: 2 and having an activity of addinga sugar molecule to an R¹ group at position 24 of a compound representedby the formula (I)” herein include a protein consisting of an amino acidsequence in which, for example, 1 to 45, 1 to 44, 1 to 43, 1 to 42, 1 to41, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 aminoacid residue is deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 2 and having an activity of adding asugar molecule to an R¹ group at position 24 of a compound representedby the formula (I). The smaller number of deletions, substitutions,insertions, and/or additions of the amino acid residues is typicallymore preferable.

Examples of such a protein include a protein having an amino acidsequence having a sequence identity of 90% or more, 91% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more,99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% ormore, or 99.9% or more with the amino acid sequence of SEQ ID NO: 2 andhaving an activity of adding a sugar molecule to an R¹ group at position24 of a compound represented by the formula (I). The larger numericalvalue of the sequence identity is typically more preferable.

Here, the “activity of adding a sugar molecule to an R¹ group atposition 24 of a compound represented by the formula (I)” means anactivity of adding a sugar to an R¹ group at position 24 of a compoundrepresented by the following formula (I).

In the formula (I), R and R¹ each independently represent H, amonosaccharide, or a disaccharide. Here, the monosaccharide is notparticularly limited.

“Monosaccharide” herein is not particularly limited; however, it may bea pentose or a hexose.

Examples of the pentose include ribose, arabinose, xylose, and lyxose,and examples of the hexose include allose, altrose, glucose, mannose,gulose, idose, galactose, and talose.

“Monosaccharide” may preferably be a hexose, more preferably a glucosemonomer (-Glc).

“Disaccharide” herein is not limited provided that it is one formed bythe binding of 2 monosaccharide molecules, and the combination of the 2monosaccharide molecules may be any combination.

The monosaccharides constituting the disaccharide may preferably be bothhexoses, and the disaccharide may more preferably be a glucose dimer(-Glc-Glc). In the disaccharide of a glucose dimer, the glucosemolecules are preferably bound by a β2,1 glycosidic bond.

The compound of the formula (I) is preferably mogrol or a mogrolglycoside.

The sugar molecule added to an R¹ group at position 24 of a compoundrepresented by the formula (I) by the protein of the present inventionis not particularly limited; however, it may be a sugar moleculeconsisting of one or more pentoses or hexoses or a combination thereof.Examples of the pentose and the hexose are as described above. The sugarmolecule is preferably a hexose, more preferably a hexose selected fromthe group consisting of glucose, mannose, and galactose. The sugarmolecule is most preferably glucose.

The activity of adding a sugar molecule to an R¹ group at position 24 ofa compound represented by the formula (I) can be verified by performingincubation in a buffer solution (for example, sodium phosphate buffer orpotassium phosphate buffer), in the neutral pH range of 6.0 to 8.0,containing 1 to 500 ng (preferably 50 to 200 ng, most preferably 100 ng)of a test protein, 0.1 to 5 mM (preferably 1 to 3 mM, most preferably 2mM) of a UDP-sugar (for example, UDP-glucose), and 0.1 to 3 mM(preferably 0.1 to 1 mM, most preferably 0.2 mM) of a substrate compound(a compound of the formula (I)) at a temperature of 20 to 40° C. for 10minutes to 5 hours (preferably 1 to 3 hours, for example, 3 hours),followed by purifying the substrate compound and then analyzing thepurified mogrol or mogrol glycosie using a known technique, such asLC-MS (Liquid Chromatography-Mass Spectrometry) analysis.

When a compound in which a sugar molecule is added to an R¹ group atposition 24 of a compound represented by the formula (I) is detected asa result of the LC-MS analysis, the test protein can be said to have theactivity of adding a sugar molecule to an R¹ group at position 24 of acompound represented by the formula (I).

The sugar addition reaction is typically completed in approximately 1minute to 12 hours. Deletion, substitution, insertion and/or addition ofone or more amino acid residues in the amino acid sequence of theprotein of the present invention is intended to mean that deletion,substitution, insertion and/or addition of one or more amino acidresidues occurs at any one or more positions in the same sequence, andtwo or more of deletion, substitution, insertion and addition may occurat the same time.

Examples of interchangeable amino acid residues are shown below. Aminoacid residues included in the same group are interchangeable with eachother. Group A: leucine, isoleucine, norleucine, valine, norvaline,alanine, 2-aminobutanoic acid, methionine, o-methylserine,t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: asparticacid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipicacid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D:lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline,4-hydroxyproline; Group F: serine, threonine, homoserine; Group G:phenylalanine, tyrosine.

Although the protein of the present invention may be obtained by beingexpressed from a polynucleotide encoding it (see “the polynucleotide ofthe present invention” described later) in appropriate host cells, itmay also be prepared by chemical synthesis methods such as Fmoc method(fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonylmethod). Alternatively, the protein of the present invention may also bechemically synthesized with peptide synthesizers commercially availablefrom Advanced Automation Peptide Protein Technologies, Perkin Elmer,Protein Technologies, PerSeptive, Applied Biosystems, SHIMADZU, etc.

2. Method for Producing Mogrol Glycoside

The present invention can easily produce a large amount of a mogrolglycoside by use of the activity of adding a sugar molecule to an R¹group at position 24 of a compound represented by the formula (I) whichthe protein has.

Accordingly, in another embodiment, the present invention provides afirst method for producing a mogrol glycoside, including a step ofadding a sugar molecule to an R¹ group at position 24 of a compoundrepresented by the following formula (I) by reacting the protein of thepresent invention, a UDP-sugar, and a compound represented by theformula (I).

R and R¹ in the formula (I) are as defined above. The compound of theformula (I) is preferably mogrol or a mogrol glycoside.

“UDP-sugar” herein is a uridine diphosphate (UDP)-bound sugar. Preferredexamples of the sugar part of the UDP-sugar include a sugar consistingof one or more pentoses or hexoses or a combination thereof. Examples ofthe pentose and the hexose are as described above. The UDP-sugar ispreferably a UDP-hexose, and the sugar is more preferably a hexoseselected from the group consisting of glucose, mannose, and galactose.The UDP-sugar is most preferably UDP-glucose.

The first method for producing a mogrol glycoside according to thepresent invention includes a step of reacting the protein of the presentinvention, a UDP-sugar, and a compound represented by the formula (I) toadd the sugar molecule to an R¹ group at position 24 of the compoundrepresented by the formula (I). The first production method of thepresent invention may further include a step of purifying the mogrolglycoside formed in the above step.

Examples of the mogrol glycoside formed by the first production methodinclude, but not limited to, mogroside IA, mogroside Ib, mogroside IE,mogroside IIA, mogroside IIA₁, mogroside IIE, mogroside III, mogrosideIIIA₁, mogroside IIIA₂, mogroside IIIE, mogroside IV, mogroside IVA,mogroside V, siamenoside I, 11-oxomogroside V, mogrol pentaglycoside, ora combination thereof.

The formed mogrol glycoside can be extracted using a suitable solvent(an aqueous solvent, such as water, or an organic solvent, such asalcohol, ether, or acetone) and purified by a known method, such asorganic solvent (e.g. ethyl acetate): water gradient, high performanceliquid chromatography (HPLC), gas chromatography, time-of-flight massspectrometry (TOF-MS), or ultra (high) performance liquid chromatography(UPLC).

3. Non-Human Transformant Highly Containing Mogrol Glycoside

The mogrol glycoside may also be formed in cells of a microorganism(e.g. E. coli or yeast), a plant, an insect, or a mammal other than ahuman using the protein of the present invention. This is because theprotein of the present invention is expected to have high activity evenin the intracellular environment since it is a Siraitiagrosvenorii-derived enzyme or its variant. In this case, the mogrolglycoside can be formed by expressing the protein of the presentinvention by introducing a polynucleotide encoding the protein of thepresent invention (see “Polynucleotide of the Invention” to be describedlater) into host cells derived, for example, from a microorganism, aplant, an insect, or a mammal other than a human, and reacting theprotein of the present invention, a UDP-sugar present in the cells, anda compound represented by the formula (I).

Accordingly, the present invention provides a non-human transformantinto which a polynucleotide selected from the group consisting of thefollowing (a) to (e) (hereinafter referred to as “polynucleotide of thepresent invention”) is introduced (hereinafter referred to as“transformant of the present invention”):

(a) a polynucleotide containing the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide encoding a protein consisting of the amino acidsequence of SEQ ID NO: 2;

(c) a protein consisting of an amino acid sequence in which 1 to 45amino acids are deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 2 and having an activity of adding asugar molecule to an R¹ group at position 24 of a compound representedby the formula (I);

(d) a polynucleotide encoding a protein having an amino acid sequencehaving 90% or more sequence identity with the amino acid sequence of SEQID NO: 2 and having the activity of adding a sugar molecule to an R¹group at position 24 of the compound represented by the formula (I); and

(e) a polynucleotide hybridizing to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 1 under highly stringent conditions, the polynucleotide encoding aprotein having the activity of adding a sugar molecule to an R¹ group atposition 24 of the compound represented by the formula (I).

The definition and specific examples of the formula (I) are as alreadydescribed, and the definition and specific examples of the sugarmolecule added to an R¹ group at position 24 of a compound representedby the formula (I) are also as described above.

“Polynucleotide” herein means DNA or RNA.

“Polynucleotide hybridizing under highly stringent conditions” hereinrefers to a polynucleotide obtained by use of, for example, a colonyhybridization method, a plaque hybridization method, or a southernhybridization method using, for example, a polynucleotide consisting ofa nucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 1 or all or part of a polynucleotide consisting of a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 2 as a probe.The hybridization method used may be a method as described, for example,in “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3,Cold Spring Harbor, Laboratory Press 2001” or “Ausubel, CurrentProtocols in Molecular Biology, John Wiley & Sons 1987-1997”.

Examples of “highly stringent conditions” herein include, but notlimited to, conditions of (1) 5×SSC, 5×Denhardt's solution, 0.5% SDS,50% formamide, and 50° C., (2) 0.2×SSC, 0.1% SDS, and 60° C., (3)0.2×SSC, 0.1% SDS, and 62° C., or (4) 0.2×SSC, 0.1% SDS, and 65° C. Inthese conditions, increasing the temperature can be expected to moreefficiently provide DNA having high sequence identity. In this regard,possible factors affecting the stringency of hybridization are aplurality of factors, such as temperature, probe concentration, probelength, ionic strength, time, and salt concentration, and one skilled inthe art can achieve similar stringency by properly selecting thesefactors.

When a commercially available kit is used for hybridization, AlkphosDirect Labelling and Detection System (GE Healthcare) can be used, forexample. In this case, according to the protocol included in the kit,incubation with a labeled probe can be carried out overnight, followedby washing the membrane with a primary washing buffer containing 0.1%(w/v) SDS under conditions of 55 to 60° C. and then detecting thehybridized DNA. Alternatively, when a probe is prepared based on anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 1 or a sequence complementary to all or part of a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 2, the labelingof the probe with digoxigenin (DIG) using a commercially availablereagent (for example, a PCR labeling mix (Roche Diagnostics K.K.))enables the detection of hybridization using DIG Nucleic Acid DetectionKit (Roche Diagnostics K.K.).

Examples of hybridizable polynucleotides other than those describedabove can include DNA having a sequence identity of 80% or more, 81% ormore, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more,87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more,99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% ormore, or 99.9% or more with DNA of SEQ ID NO: 1 or DNA encoding theamino acid sequence of SEQ ID NO: 2 when calculated with a homologysearch software, such as FASTA or BLAST, using default parameters.

The sequence identity of an amino acid sequence or a nucleotide sequencecan be determined using FASTA (Science 227 (4693): 1435-1441 (1985)) oralgorithm BLAST (Basic Local Alignment Search Tool) by Karlin andAltschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl.Acad. Sci. USA 90: 5873, 1993). Programs called balstn, blastx, blastp,tblastn, and tblastx based on the algorithm of BLAST have been developed(Altschul S F, et al.: J. Mol. Biol. 215: 403, 1990). In analyzing anucleotide sequence using blastn, the parameters are set, for example,to score=100 and wordlength=12. In analyzing an amino acid sequenceusing blastp, the parameters are set, for example, to score=50 andwordlength=3. In using BLAST and Gapped BLAST programs, the defaultparameters of the respective programs are used.

The polynucleotide of the present invention described above can beobtained by a known genetic engineering technique or a known synthesistechnique.

The polynucleotide of the present invention is preferably introducedinto a host in a state inserted into a suitable expression vector.

The suitable expression vector is typically configured to include:

(i) a promoter capable of transcription in host cells;

(ii) the polynucleotide of the present invention, bound to the promoter;and

(iii) an expression cassette containing signals functioning in the hostcells for the transcription termination and polyadenylation of an RNAmolecule, as constituents.

Examples of a method for preparing the expression vector include, butnot particularly limited to, a method using a plasmid, a phage, or acosmid.

The specific type of the vector is not particularly limited, and avector capable of being expressed in host cells is properly selected.Specifically, a promoter sequence is properly selected to reliablyexpress the polynucleotide of the present invention, depending on thetype of host cells, and a vector in which the promoter sequence and thepolynucleotide of the present invention are incorporated into any ofvarious plasmids or the like may be used as the expression vector.

The expression vector of the present invention contains an expressioncontrol region (for example, a promoter, a terminator, and/or areplication origin), depending on the type of a host into which thevector is to be introduced. A conventional promoter (for example, trcpromoter, tac promoter, or lac promoter) is used as an expression vectorfor bacteria; examples of a promoter for yeast include glyceraldehyde3-phosphate dehydrogenase promoter and PH05 promoter; and examples of apromoter for filamentous bacteria include amylase and trpC. Examples ofa promoter for expressing a target gene in plant cells include 35S RNApromoter of cauliflower mosaic virus, rd29A gene promoter, rbcSpromoter, and mac-1 promoter in which an enhancer sequence in the 35SRNA promoter of cauliflower mosaic virus is added 5′ to a mannopinesynthase promoter sequence derived from Agrobacterium. Examples of apromoter for an animal cell host include viral promoters (for example,SV40 initial promoter and SV40 late promoter).

The expression vector preferably contains at least one selection marker.As such a marker, an auxotrophic marker (ura5, niaD), a drug-resistancemarker (hygromycin, Zeosin), a geneticin resistance gene (G418r), acopper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA,vol. 81, p. 337, 1984), a cerulenin resistance gene (fas2m, PDR4) (inJunji Inokoshi et al., Seikagaku (Biochemistry), vol. 64, p. 660, 1992and Hussain et al., Gene, vol. 101, p. 149, 1991, respectively), or thelike can be used.

The method for preparing (method for producing) the transformant of thepresent invention is not particularly limited; however, examples thereofinclude a method involving introducing an expression vector containingthe polynucleotide of the present invention into a host fortransformation.

The transformant of the present invention is expected to contain amogrol glycoside at a high content. The host cell used fortransformation is not particularly limited and various known cells canpreferably be used. Examples of the host cell include bacteria, such asEscherichia coli, yeast (Saccharomyces cerevisiae as budding yeast orSchizosaccharomyces pombe as fission yeast), plant cells, and cells ofanimals other than humans.

The host cell is not limited to one capable of forming a compoundrepresented by the formula (I); however, the host cell is preferably ahost cell capable of forming a compound represented by the formula (I).Here, the host cell may be one subjected to recombination with a knowngene to be in a natural state, for example, to be capable of forming acompound represented by the formula (I).

Examples of a known gene encoding an enzyme contributing to thesynthesis of a compound represented by the formula (I) include, but notlimited to, a mogrol or mogroside synthesis-associated gene as describedin cucurbitadienol synthase (M. Shibuya et al./Tetrahedron 60 (2004)6995-7003).

When the host cell is one incapable of forming a compound represented bythe formula (I), a compound represented by the formula (I) or a plantextract containing the compound can be added as a substrate to a culturesystem of the transformant obtained by introducing the gene of thepresent invention into the host cell to produce a mogrol glycosidewithout introducing a gene encoding an enzyme contributing to thesynthesis of a compound represented by the formula (I).

Suitable culture media and conditions for the above host cells are wellknown in the art. The living organism to be transformed is notparticularly limited; examples thereof include various microorganisms,plants, or animals other than humans exemplified in the above hostcells.

As a method for transforming host cells, commonly used known methods canbe used. The transformation can be carried out by, for example, but notlimited to, an electroporation method (Mackenxie, D. A. et al., Appl.Environ. Microbiol., vol. 66, p. 4655-4661, 2000), a particle deliverymethod (Japanese Patent Application Laid-Open No. 2005-287403), aspheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978),a lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983), or amethod as described in Methods in yeast genetics, 2000 Edition: A ColdSpring Harbor Laboratory Course Manual).

For other common molecular biological techniques, one may refer to, forexample, “Sambrook & Russell, Molecular Cloning: A Laboratory ManualVol. 3, Cold Spring Harbor Laboratory Press 2001”, “Methods in YeastGenetics, A laboratory manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.”).

The culture of the transformant thus obtained enables the accumulationof a mogrol glycoside in the transformant. As described above, acompound of the formula (I) or a plant extract containing the compoundcan also be added as a substrate to a culture system of the transformantto promote the production of the mogrol glycoside. The accumulatedmogrol glycoside can be extracted and purified to provide an intendedmogrol glycoside.

Thus, the present invention provides a second method for producing amogrol glycoside, comprising using the transformant of the presentinvention. Suitable culture media and conditions are well known in theart. The method for extracting and purifying the mogrol glycoside is asalready described.

The mogrol glycoside is not particularly limited; however, the glycosidemay preferably be one selected from the group consisting of mogrosideIA, mogroside Ib, mogroside IE, mogroside IIA, mogroside IIA₁, mogrosideIIE, mogroside III, mogroside IIIA₁, mogroside IIIA₂, mogroside IIIE,mogroside IV, mogroside IVA, mogroside V, siamenoside I, 11-oxomogrosideV, mogrol pentaglycoside, and a combination thereof.

In one aspect of the present invention, the transformant can be a planttransformant. The plant transformant according to the present embodimentis obtained by introducing a recombinant vector containing thepolynucleotide according to the present invention into a plant in such away that a polypeptide encoded by the polynucleotide can be expressed.

When using a recombinant expression vector, the recombinant expressionvector used for the transformation of a plant body is not particularlylimited provided that it is a vector capable of expressing thepolynucleotide according to the present invention in the plant. Examplesof such a vector include a vector having a promoter constitutivelycausing the expression of a polynucleotide in plant cells, and a vectorhaving a promoter inducibly activated by an external stimulus.

Examples of the promoter constitutively causing the expression of apolynucleotide in plant cells include 35S RNA promoter of cauliflowermosaic virus, rd29A gene promoter, rbcS promoter, and mac-1 promoter.

Examples of the promoter inducibly activated by an external stimulusinclude a mouse mammary tumor virus (MMTV) promoter, atetracycline-responsive promoter, a metallothionein promoter, and a heatshock protein promoter.

The plant to be transformed according to the present invention means anyof the whole plant body, a plant organ (e.g., leaf, petal, stem, root,or seed), a plant tissue (e.g., cuticle, phloem, parenchyma, xylem,bundle, palisade tissue, or spongy tissue), a plant cultured cell, anyof various forms of plant cells (e.g., a suspension cultured cell), aprotoplast, a leaf segment, and a callus. The plant used fortransformation is not particularly limited, and may be any of plantsbelonging to Liliopsida and Magnoliopsida.

Transformation methods well known to those of ordinary skill in the art(for example, an Agrobacterium method, a gene gun method, a PEG method,and an electroporation method) are used for the introduction of a geneinto a plant. For example, a method through Agrobacterium and a methodinvolving direct introduction into plant cells are well-known. When theAgrobacterium method is used, a transformed plant can be obtained byintroducing a constructed expression vector for a plant into a suitableAgrobacterium strain (for example, Agrobacterium tumefaciens) andcausing this strain to infect an aseptically cultured leaf discaccording to a leaf disc method (Hirofumi Uchimiya, “Shokubutsu IdenshiSosa Manyuaru (Plant genetic engineering manual)” (1990) p. 27-31,Kodansha Scientific, Tokyo, or the like). The method of Nagel et al.(Micribiol. Lett., 67: 325 (1990)) may be also used. This method is amethod which involves first introducing, for example, an expressionvector into Agrobacterium and then introducing the transformedAgrobacterium into a plant cell or a plant tissue by a method asdescribed in Plant Molecular Biology Manual (Gelvin, S. B. et al.,Academic Press Publishers). Here, “plant tissue” includes a callusobtained by the culture of plant cells. When transformation is carriedout using the Agrobacterium method, a binary vector (pBI121, PZP202, orthe like) can be used.

As the method involving directly introducing a gene into a plant cell ora plant tissue, an electroporation method and a particle gun method areknown. When the particle gun is used, a plant body, a plant organ, or aplant tissue may be directly used, may be used after preparing thesection thereof, or may be used by preparing a protoplast. The specimenthus prepared can be treated using a gene introduction device (forexample, PDS-1000 (BIO-RAD Co., Ltd.)). Treatment conditions varydepending on the plant or the specimen; however, they are typically apressure of on the order of 450 to 2,000 psi and a distance of on theorder of 4 to 12 cm.

The gene-introduced cell or plant tissue is first selected by drugresistance, such as hygromycin resistance, and then regenerated into aplant body by an ordinary method. The regeneration of the plant bodyfrom the transformed cell can be carried out by a method known to thoseof ordinary skill in the art, depending on the type of the plant cell.

When cultured plant cells are used as a host, transformation is carriedout by introducing a recombinant vector into the cultured cells by thegene gun or electroporation method or the like. The callus, shoot, hairyroot, or the like obtained as a result of transformation can be directlyused for cell culture, tissue culture or organ culture and can beregenerated into a plant body using a heretofore known plant tissueculture method, for example, by administering a suitable concentrationof a plant hormone (e.g. auxin, cytokinin, gibberellin, abscisic acid,ethylene, or brassinoride).

Whether or not the polynucleotide of the present invention has beenintroduced into a plant can be confirmed using a PCR method, a Southernhybridization method, a Northern hybridization method, or the like. Forexample, DNA is prepared from a transformed plant, and primers specificfor the DNA are designed to perform PCR. PCR can be carried out underthe same conditions as those used for preparing the above plasmid.Thereafter, the amplified product can be subjected to agarose gelelectrophoresis, polyacrylamide gel electrophoresis, or capillaryelectrophoresis, stained with ethidium bromide, SYBR Green solution orthe like, and detected as a one band to confirm transformation. Theamplified product can also be detected by performing PCR using primerslabeled with a fluorescent dye or the like in advance. In addition, amethod can be adopted which involves binding the amplified product to asolid phase, such as a microplate, and confirming the amplified productby fluorescence or enzyme reaction.

Once a transformed plant body in which the polynucleotide of the presentinvention is incorporated in the genome is obtained, an offspring can beobtained by the sexual reproduction or asexual reproduction of the plantbody. From the plant body or its offspring or their clones, for example,a seed, a fruit, a cutting, a tuber, a root tuber, a stump, a callus, ora protoplast is obtained, and the plant body can be mass produced basedon them. Thus, also included in the present invention is a plant bodyinto which the polynucleotide according to the present invention isexpressively introduced, an offspring of the plant body, having the sameproperties as those of the plant body, or tissues derived therefrom.

Transformation methods for various plants have already been reported.Examples of the transformant plant according to the present inventioninclude Solanaceae plants (for example, eggplant, tomato, peppers,potato, tobacco, datura, nightshade, petunia, Calibrachoa, Nierembergia,etc.), Leguminosae plants (for example, soybean, azuki bean, peanut,kidney bean, broad bean, lotus, etc.), Rosaceae plants (for example,strawberry, plum, cherry, rose, blueberry, blackberry, bilberry, blackcurrant, raspberry, Rubus suavissimus, etc.), Caryophyllaceae plants(carnation, gypsophila, etc.), Asteraceae plants (chrysanthemum,gerbera, sunflower, daisy, stevia, etc.), Orchidaceae plants (orchid,etc.), Primulaceae plants (cyclamen, etc.), Gentianaceae plants(lisianthus, gentian, etc.), Iridaceae plants (freesia, iris, gladiolus,etc.), Scrophulariaceae plant (snapdragon, Torenia, etc.), Crassulaceae(kalanchoe), Liliaceae plants (lily, tulip, etc.), Convolvulaceae plants(morning glory, ipomoea cairica, moonflower, sweet potato, cypress vine,evolvulus, etc.), Hydrangeaceae plants (hydrangea, deutzia, etc.),Cucurbitaceae plants (cucumber, bitter gourd, pumpkin, loofah, Siraitiagrosvenorii, bottle gourd, etc.), Geraniaceae plants (Pelargonium,geranium, etc.), Oleaceae plants (forsythia, etc.), Vitaceae plants (forexample, grape, etc.), Theaceae plants (camellia, tea, etc.), Gramineaeplants (for example, rice, barley, wheat, oat, rye, corn, millet,Japanese millet, kaoliang, sugar cane, bamboo, wild oat, finger millet,sorghum, wild rice, pearl barley, pasture grass, etc.), Moraceae plants(mulberry, paper mulberry, rubber tree, hemp, etc.), Rubiaceae plant(Coffea, gardenia, etc.), Fagaceae plants (oak, beech, Kashiwa oak,etc.), Pedaliaceae plants (sesame, etc.), Rutaceae plants (for example,bitter orange, citron, satsuma mandarin, Japanese pepper) andBrassicaceae plants (red cabbage, ornamental cabbage, Japanese radish,Arabidopsis thaliana, Brassica rapa L. var. nippo-oleifera, cabbage,broccoli, cauliflower, etc.), Labiatae (salvia, beefsteak plant,lavender, skull cap, etc.), and Cannabaceae (hop, etc.). Particularlypreferred examples of the plant to be transformed used are plants knownto biosynthesize various glycosides using mogrol or a mogrol glycosideas a sugar receptor; examples of such plants include Siraitiagrosvenorii and Rubus suavissimus.

The plant body transformed with the polynucleotide of the presentinvention (hereinafter referred to as “plant of the present invention”or “plant body of the present invention”) can produce a large amount ofa mogrol glycoside compared to its wild type one if it has a suitablesubstrate or if a suitable substrate is externally added.

The plant of the present invention can easily provide a complete plantbody by the cultivation of seeds, cuttings, bulbs, or the like of theplant of the present invention.

Thus, included in the plant of the present invention are included thewhole plant body, plant organs (e.g., leaf, petal, stem, root, seed, andbulb), plant tissues (e.g., cuticle, phloem, parenchyma, xylem, bundle,palisade tissue, and spongy tissue), plant cultured cells, various formsof plant cells (e.g., suspension cultured cells), protoplasts, leafsegments, calluses, and the like.

4. Transformant Extract and Use Thereof

In another embodiment, the present invention provides an extract of theabove transformant. The transformant of the present invention has a highcontent of a mogrol glycoside compared to its wild type one if it has asuitable substrate or if a suitable substrate is externally added; thus,the mogrol glycoside is probably contained in a high concentration inits extract.

The extract of the transformant of the present invention can be obtainedby crushing the transformant using, for example, glass beads, ahomogenizer, or a sonicator, subjecting the crushed product tocentrifugal treatment, and recovering the supernatant. In addition, afurther extraction step may be provided using the above-described methodfor extracting a mogrol glycoside.

The extract of the transformant of the present invention can be used inapplications of, for example, the production of a food, a pharmaceuticalproduct, or an industrial raw material and the like according to anordinary method.

In another embodiment, the present invention provides a food, amedicine, and an industrial raw material (a raw material for foods orthe like) which contain the extract of the transformant of the presentinvention. The food, medicine, and industrial raw material containingthe extract of the transformant of the present invention are preparedaccording to ordinary methods. Thus, the food, medicine, industrial rawmaterial, or the like containing the extract of the transformant of thepresent invention contains a mogrol glycoside formed using thetransformant of the present invention.

The dosage form of the pharmaceutical product (composition) of thepresent invention is not particularly limited and may be any dosageform, such as solutions, pastes, gels, solids, or powders. Thepharmaceutical composition of the present invention can be used in bathagents, hair tonics, skin beauty liquids, sunscreen agents, and the likein addition to medicines for external use, such as oils, lotions,creams, emulsions, gels, shampoos, hair rinses, hair conditioners,enamels, foundations, lipsticks, face powders, packs, ointments,powders, dentifrices, aerosols, and cleansing foams.

The pharmaceutical composition of the present invention may furthercontain other pharmaceutically active ingredients (for example, ananti-inflammatory ingredient) or auxiliary components (for example, alubricating component and a carrier component), if necessary.

Examples of the food of the present invention include a dietarysupplement, a health food, a functional food, an infant food, and ageriatric food. The food herein is a solid, a fluid, a liquid, or amixture thereof, and is a generic term applied to ingestible materials.

The dietary supplement refers to a food in which specific nutritionalingredients are enriched. The health food refers to a food which ishealthy or considered to be good for the health, and includes a dietarysupplement, a natural food, and a diet food. The functional food refersto a food for replenishing nutritional ingredients performing bodycontrol functions, and is the same meaning as a food for specifiedhealth use. The infant food refers to a food for giving to children upto about 6 years old. The geriatric food refers to a food treated so asto be easily digested and absorbed compared to the untreated food.

The food of the present invention uses a mogrol glycoside, which is acalorie-less sweetener. Thus, the food of the present invention has themerit of being low in calories and contributing to health promotion orhealth maintenance.

Examples of the form of these foods may be agricultural food products,such as bread, noodles, pasta, rice, confectionery (cakes, ice cream,ice candy, doughnut, baked cakes, candy, chewing gum, gummi, tabletconfectionery, and Japanese sweets (e.g., dumplings and buns with abean-jam filling)) and tofu and its processed products; fermented foods,such as sake, medicinal beverage, sweet sake, vinegar, soy sauce, andmiso; livestock products, such as yogurt, ham, bacon, and sausage; seafoods, such as boiled fish paste, fried fish-meat paste, and boiled flatfish cake; and juice drinks, soft drinks, sports beverages, alcoholicbeverages, teas, or seasonings.

5. Method of Screening for Plant Having High Mogrol Glycoside Content

The present invention provides a method of screening for a plant havinga high mogrol glycoside content. Specifically, the method includes thefollowing (1) to (3) steps:

(1) a step of extracting mRNA from a test plant;

(2) a step of hybridizing the mRNA or cDNA prepared from the mRNA with apolynucleotide capable of hybridizing with a polynucleotide consistingof a nucleotide sequence complementary to the polynucleotide of thepresent invention under highly stringent conditions; and

(3) a step of detecting the above hybridization.

The above step (1) can be carried out by extracting mRNA from a testplant. The part of a test plant from which mRNA is extracted is notparticularly limited; however, it is preferably a petal. When mRNA hasbeen extracted, cDNA may be prepared from the mRNA by reversetranscription.

The step (2) can be carried out by hybridizing the extracted mRNA with apolynucleotide or oligonucleotide consisting of a nucleotide sequencecomplementary to the polynucleotide of the present invention as a probeor primer under highly stringent conditions. The highly stringentconditions are as already described. The polynucleotide oroligonucleotide preferably has a length of 5 to 500 bp, more preferably10 to 200 bp, still more preferably 10 to 100 bp. The polynucleotide oroligonucleotide can be easily synthesized using any of various automatedsynthesizers (for example, AKTA oligopilot plus 10/100 (GE HealthCare)), or can also be synthesized under commission to a third-partyorganization (for example, Promega or Takara) or the like.

When the polynucleotide consisting of a nucleotide sequencecomplementary to the polynucleotide of the present invention is used asa probe in the step (2), the step (3) can be carried out by ahybridization detection method, such as conventional Southern blotting,Northern blotting (Sambrook, Fritsch and Maniatis, “Molecular Cloning: ALaboratory Manual” 2nd Edition (1989), Cold Spring Harbor LaboratoryPress), microarray (Affymetrix Co., Ltd.; see U.S. Pat. Nos. 6,045,996,5,925,525, and 5,858,659), TaqMan PCR (Sambrook, Fritsch and Maniatis,“Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold SpringHarbor Laboratory Press), or fluorescent in situ hybridization (FISH)(Sieben V. J. et al., (2007-06), IET Nanobiotechnology 1(3): 27-35).When the polynucleotide consisting of a nucleotide sequencecomplementary to the polynucleotide of the present invention is used asa primer in the step (2), the hybridization detection in the step (3)can be carried out by performing PCR amplification reaction andanalyzing the resultant amplified product by electrophoresis orsequencing (Sambrook, Fritsch and Maniatis, “Molecular Cloning: ALaboratory Manual” 2nd Edition (1989), Cold Spring Harbor LaboratoryPress).

A plant body for which more hybridization is detected can be said toexpress a larger amount of a protein having an activity of adding asugar molecule to an R¹ group at position 24 of a compound representedby the following formula (I) than that for other plant bodies, and thusis expected to have a high mogrol glycoside content.

EXAMPLES

The present invention will be more specifically described below withreference to Examples. However, the scope of the present invention isnot intended to be limited to such Examples.

Example 1: Isolation of Candidate Gene of Mogrol Glycosyltransferase

The molecular biological technique used in this Example was according tothe method described in Molecular Cloning (Sambrook et al., Cold SpringHarbor Laboratory Press, 2001) until detailed otherwise.

Tanscriptome analysis (RNA-sequencing) for the fruit of Siraitiagrosvenorii using a next-generation sequencer (NGS) has been published(Tang, Q. et al. (2011) MC Genomics 12: 343); for the sequenceinformation, sequences having high identity to the knownglycosyltransferase were selected by identity analysis using blastn.

Example 2: Functional Analysis of Candidate Gene

Based on the sequence information of SgUGT74G1_943, one of the resultantcandidate genes, (SEQ ID NO: 1), SgUGT74G1_943 gene was artificiallysynthesized in ThermoFiosher Co., Ltd. The resultant synthetic genefragment was incorporated into the restriction enzyme sites NdeI andBamHI of an E. coli expression vector, pET15b (Novagen Co., Ltd.), anddesigned so that HisTag is fused to the N-terminal. The vector wastransformed into E. coli (BL21_DE3) according to an ordinary method, anda recombinant protein was expressed using the overnight express (TM)autoinduction system from Millipore Co., Ltd. according to the protocolprovided by the manufacturer. The expression of the recombinant proteincould be confirmed as a band of around 50 kDa as deduced by CBB stainingand Western blot analysis with a mouse anti-HiSTag antibody (FIG. 2).FIG. 2 shows the results of expression analysis of the recombinantprotein by Coomassie brilliant blue staining (CBB: left Figure) andWestern blotting analysis (WB: right Figure); in the figure, Mrepresents a size marker, NC represents negative control (pET15b/BL21(strain DE3)), and S represents a sample (SgUGT74G-943/pEt15b/BL21(strain DE3)).

Purification was carried out using the HisTag of the recombinant proteinaccording to an ordinary method, and glucose transfer activity formogrol prepared by hydrolyzing a Siraitia grosvenorii extract (Takemoto,T. et al. (1983) YAKUGAKU ZASSHI 103(11), 1167-1173) was evaluated. Theenzyme reaction composition was 0.2 mM mogrol as a glucose acceptor, 2mM UDP-glucose as a glucose donor, and 50 mM calcium phosphate buffer(pH 7.5); an enzyme solution was added thereto, which was then reactedat 30° C. for 3 hours; and the product was analyzed using LC-IT-TOF-MS(Shimadzu Corporation).

As a result, a new product was observed in addition to mogrol in thereaction solution of SgUGT74G_943 enzyme (FIG. 3). FIG. 3A is an LC-MSanalysis chart of the reaction solution between SgUGT74G_943 and mogrol;the red line represents a Na adduct ion of mogrol having one molecule ofglucose added corresponding to m/z=661, the pink line represents a Naadduct ion of mogrol corresponding to m/z=499, the blue line representsa proton adduct of mogrol corresponding to m/z=477, and the black linerepresents TIC (total ion chromatography of positive ions).

Aside from mogrol as a substrate, no peak of a product was observed inthe reaction solution of the vector control (FIG. 3B). In the LC-MSanalysis chart for the reaction solution of the negative controlexpressing an empty vector in FIG. 3B, ion peak derived from mogrol wasonly observed. The product was concluded to be mogrol monoglycoside inwhich one molecule of glucose was added by SgUGT74_943, from its massand retention time. The sugar-binding site was found to be position 24of mogrol from comparison with past references (Ukiya, M., et al. (2002)J. Agric. Food. Chem. 50, 6710-6715 and Takemoto, T. et al. (1983)YAKUGAKU ZASSHI 103(11), 1167-1173), which used nuclear magneticresonance (NMR).

Example 3: Activity Comparison with Known Enzyme

It has heretofore been known that a steviol glycosyltransferase derivedfrom stevia can glycosylate mogrol (WO2013/076577). Then, the mogrolglycosylation activity of Siraitia grosvenorii-derived SgUGT74-943 wascompared with the same activities of UGT85C2 and UGT73E1 of stevia.

Stevia-derived UGT85C2 and UGT73E1 were expressed in the same manner asin the above method. The product amount was measured under the aboveenzyme conditions to determine relative activity by dividing it by theexpressed protein amount. As a result, the relative activities ofstevia-derived UGT85C2 and UGT73E1 when the activity of SgUGT74-943 wasset to 1 were 0.35 and 0.22, respectively. The glycosylation activity ofSiraitia grosvenorii-derived SgUGT74-943 was confirmed to be nearly 3times higher than those of stevia-derived UGT85C2 and UGT73E1 (FIG. 4).

INDUSTRIAL APPLICABILITY

The gene of an enzyme glycosylating position 24 of mogrol was found outfrom Siraitia grosvenorii. The present invention can be used not onlyfor such breeding as to increase the mogrol glycoside content using thegene as an index but also for the study of environmental conditions forincreasing the mogrol glycoside content in cultivation and inpost-harvest. In addition, it can be used as a tool, such as an externalenzyme, for the production of a useful substance.

We claim:
 1. A polynucleotide selected from: (a) a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotidecomprising a heterologous regulatory element operably linked to apolynucleotide sequence encoding a protein consisting of the amino acidsequence of SEQ ID NO: 2; (c) a polynucleotide encoding a proteinconsisting of an amino acid sequence in which 1 to 45 amino acids aredeleted, substituted, inserted, and/or added in the amino acid sequenceof SEQ ID NO: 2 and having an activity of adding a sugar molecule to anR¹ group at position 24 of a compound represented by the formula (I);and (d) a polynucleotide hybridizing to a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of SEQ IDNO: 1 under highly stringent conditions, the polynucleotide encoding aprotein having the activity of adding a sugar molecule to an R¹ group atposition 24 of the compound represented by formula (I);

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide.
 2. A protein consisting of an amino acid sequence in which1 to 45 amino acids are deleted, substituted, inserted, and/or added inthe amino acid sequence of SEQ ID NO: 2 and having an activity of addinga sugar molecule to an R1 group at position 24 of a compound representedby formula (I)

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide.
 3. A vector comprising the polynucleotide of claim
 1. 4. Anon-human transformant comprising the polynucleotide of claim
 1. 5. Thenon-human transformant of claim 3, which is a plant or a microorganism.6. The non-human transformant of claim 3, wherein the polynucleotide isinserted into an expression vector.
 7. A method comprising culturing orcultivating the non-human transformant of claim 4 to produce the proteinconsisting of the amino acid sequence of SEQ ID NO:
 2. 8. The method ofclaim 7, further comprising: producing a mogrol glycoside by reactingthe protein consisting of the amino acid sequence of SEQ ID NO: 2produced by the non-human transformant with a UDP-sugar and a compoundrepresented by formula (I):

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide.
 9. A method of producing a mogrol glycoside, comprising:reacting, outside of a Cucurbitaceous plant, a protein consisting of theamino acid sequence of SEQ ID NO: 2 with a UDP-sugar and a compoundrepresented by formula (I):

where R and R¹ each independently represent H, a monosaccharide, or adisaccharide.
 10. A method comprising: providing the non-humantransformant of claim 3 or a culture thereof, and obtaining an extractof the non-human transformant or of the culture of the non-humantransformant.
 11. A method comprising: providing an extract of thenon-human transformant of claim 3 or an extract of a culture of thenon-human transformant of claim 3, adding the extract to a raw materialof a food, a pharmaceutical preparation or an industrial raw material,and preparing a food, a pharmaceutical preparation or an industrial rawmaterial.
 12. The method of claim 8, wherein the UDP-sugar is aUDP-hexose.
 13. The method of claim 8, wherein the sugar molecule isselected from the group consisting of UDP-glucose, UDP-mannose, andUDP-galactose.
 14. The method of claim 8, wherein R and R¹ is eachindependently H or a sugar residue of a glucose monomer or a glucosedimer.
 15. The method of claim 8, wherein the compound represented byformula (I) is mogrol or a mogrol glycoside.
 16. The method of claim 8,wherein the mogrol glycoside is mogroside IA, mogroside Ib, mogrosideIE, mogroside IIA, mogroside IIA1, mogroside IIE, mogroside III,mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside IV,mogroside IVA, mogroside V, siamenoside I, 11-oxomogroside V, mogrolpentaglycoside, or a combination thereof.
 17. The method of claim 9,wherein the mogrol glycoside is mogroside IA, mogroside Ib, mogrosideIE, mogroside IIA, mogroside IIA1, mogroside IIE, mogroside III,mogroside IIIA1, mogroside IIIA2, mogroside IIIE, mogroside IV,mogroside IVA, mogroside V, siamenoside I, 11-oxomogroside V, mogrolpentaglycoside, or a combination thereof.
 18. The method of claim 11,wherein the food is a beverage.
 19. The method of claim 11, wherein thefood is selected from a juice drink, a soft drink, a sports beverage, analcoholic beverage, a tea, a vinegar, and a soy sauce.